Infectious prions are thought to be composed entirely of an abnormally folded conformer (PrPSc) of the cellular prion protein (PrPC). During prion propagation, PrPSc appears to serve as a self-replicating template, specifically sequestering PrPC and driving conversion of the docked substrate into additional PrPSc. Reproducible differences in incubation period, clinical symptoms and patterns of PrPSc deposition associated with different prion strains argue that prion replication is a remarkably high-fidelity, conformation-dependent process. However, although PrPC-PrPSc interactions are clearly central to the formation of nascent prion infectivity, very little is known about how these divergent conformers align during assembly of the prion replicative complex. We have shown that antibodies binding to specific regions of PrPC potently inhibit prion replication. Here we hypothesize that PrPC epitopes bound by the inhibitory antibodies form key components of one face of the PrPC-PrPSc heterocomplex, and therefore possess intrinsic affinity for PrPSc. In support of this idea, two such PrP epitopes have been grafted into an antibody scaffold, imparting upon the recipient molecule specific recognition of disease-associated PrP conformers (PrPSc and PrP27-30). The purpose of this application is to exploit our PrP motif-grafting approach to create a comprehensive panel of PrPSc-specific reagents that thoroughly and systematically map the PrPC-PrPSc binding interaction. It is proposed that antibodies recognizing infectious human, cervid and mouse prions will be generated. Sequential rounds of mutagenesis and competitive selection will be used to evolve the binding kinetics of the PrPSc-reactive antibodies to the picomolar range. In addition, the ability of these novel reagents to inhibit prion replication will be investigated. The results of this program will provide unique insight into prion replication and yield invaluable molecular tools that will find extremely broad application in the study of prion biology.